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. 2004 Feb 15;18(4):423–434. doi: 10.1101/gad.285504

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Figure 7. — mTOR controls the nucle(ol)ar localization of TIF-IA. (A) Rapamycin treatment mediates translocation of TIF-IA into the cytoplasm. HeLa cells expressing Flag-tagged TIF-IA were incubated with 20 nM rapamycin for 2 h and immunostained with Cy3-conjugated anti-Flag antibodies. Fluorescence and merged images with phase contrast from the same view are shown. (B) TIF-IA accumulates in the cytoplasm after rapamycin treatment. The levels of TIF-IA and Pol I (RPA116) were analyzed by Western blotting in whole-cell extracts (lanes 1,2), nuclear extract (lanes 3,4), and the respective S-100 fraction (lanes 5,6) from untreated or rapamycin-treated FM3A cells. (C) Rapamycin-treatment inactivates both nuclear and cytoplasmic TIF-IA. Transcription reactions contained 50 μg of nuclear extract from growing (lanes 1–3) or rapamycin-treated (lanes 4–6) cells. The assays were supplemented with 50 μg of the respective S-100 fraction (lanes 2,5) or 3 ng of cellular TIF-IA (lanes 3,6).